Phospholipases A2 are a family of enzymes that hydrolyze membrane phospholipids to generate free fatty acids and lysophospholipids. They are involved in a wide range of processes such as inflammation, phospholipid remodeling, cell proliferation and apoptosis. Using group V secretory PLA2 (sPLA2)-null mouse peritoneal macrophages we showed that group V sPLA2 amplifies the action of cytosolic PLA2alpha (cPLA2 alpha) in regulating eicosanoid generation after zymosan phagocytosis. Our recent study with group V sPLA2-null macrophages suggests that group V sPLA2 has a unique role in regulating phagocytosis, a function that is not shared with cPLA2alpha or group IIA sPLA2. Furthermore, our preliminary data show that group V sPLA2-null macrophages have attenuated phagocytosis of red blood cells opsonized with IgG or with complement and of Candida albicans. However, little is known regarding the mechanism/s of action of group V sPLA2 and how it regulates phagocytosis. We hypothesize that group V sPLA2 regulates the early steps of phagocytosis and that this function is related to its particular structural characteristics. Therefore in specific Aim 1 we will examine binding of phagocytic particles, phosphorylation of non-receptor tyrosine kinases, and activation of members of the Rho family of GTPases (molecules that are central to phagocytosis) in group V sPLA2-null and wild type peritoneal macrophages in response to phagocytic stimuli. Specific Aim 2 will test the hypothesis that group V sPLA2 is critical for the immune response to specific pathogens such as Candida albicans, which triggers the same cell surface receptors as zymosan and IgG-opsonized Klebsiella pneumoniae. To this purpose we will evaluate the ability of group V sPLA2-null macrophages to phagocytose and kill Candida albicans, non opsonized and IgG-opsonized Klebsiella pneumoniae and the mechanism/s of the putative action of group V sPLA2. We will extend these studies to an in vivo model of infection with C. albicans and K. pneumoniae. Lastly, in specific Aim 3 we will examine the relationship of the structure of group V sPLA2 to its function. We will use site-directed mutagenesis and adenoviral vectors to examine phagocytosis in group V sPLA2-null macrophages reconstituted with group V sPLA2 mutants that (i) lack interfacial binding to PC, (ii) can not bind to proteoglycan, or (iii) lack catalytic activity. Relevance. Phagocytosis is the very early step in innate and acquired immune responses such as host defense against pathogens and antigen presentation. Infectious diseases are still the major cause of death in the world. Understanding how group V sPLA2 regulates phagocytosis will help to better understand the mechanisms of allergic, immunologic, and infectious diseases in which phagocytosis has a leading role, and to develop better means of preventing, diagnosing, and treating these illnesses.